Hospital Practice
ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
The Cystic Fibrosis Gene: Isolation and Significance Francis S. Collins, John R. Riordan & Lap-Chee Tsui To cite this article: Francis S. Collins, John R. Riordan & Lap-Chee Tsui (1990) The Cystic Fibrosis Gene: Isolation and Significance, Hospital Practice, 25:10, 47-57, DOI: 10.1080/21548331.1990.11704019 To link to this article: http://dx.doi.org/10.1080/21548331.1990.11704019
Published online: 17 May 2016.
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The Cystic Fibrosis Gene: Isolation and Significance and LAP-CHEE University of Michigan and University qf Toronto
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FRANCIS S. COLLINS, JOHN R RIORDAN,
TSUI
The identification and cloning of the CF gene, which codes for a membrane protein that appears to regulate transmembrane ion transport, may lead to an understanding of the basic defect in the disease and to more effective treatment. More broadly, the cloning of the CF gene provides a fast start in the international effort to clone and map the entire human genome.
The search for the cystic fibrosis gene was a long and arduous process that required considerable time, energy, and money. Two research groups in Ann Arbor, Mich., and Toronto, working in close collaboration, finally identified the gene in September 1989-almost a decade after the search began. Almost as soon as the gene was in hand, improvements in prenatal diagnosis and carrier screening became possible. The therapeutic benefits that may follow from cloning the gene, however, are still probably some years away. The expectation is that they will arise from a better understanding of the basic biologic problem and perhaps involve gene therapy. The identification of the CF gene represented a solution to a challenging problem, one that has given additional momentum to the efforts to map and sequence the human genome. That genome is usually described as a library of 3 billion characters or as a blueprint for building human beings. The metaphors imply that if one gains access to the library and knows how to solve the code, one can read the approximately 100,000 human genes like an owner's manual. The manual is not very "user-friendly,'' however. Genes (DNA} code for RNA. which codes for proteins. Where those proteins go, what they do, how they interact with other proteins or fail to interact are exceedingly difficult biochemical questions. The answers may be written in the genes but are not directly interpretable with current knowledge. With this perspective kept in mind, we can now proceed to review the work involved in identification of the CF gene.
Cystic fibrosis affects an estimated 30,000 young Americans, and an additional 2,000 babies are born with the disease every year. It is the most common severe autosomal recessive disease in whites. Antibiotic therapy of secondary bacterial infections, improved chest physical therapy. and better nutrition have increased life expectancy in CF patients. but little progress has been made in treating the basic defect. The disturbing prognosis remains: Without dramatic clinical advances, most patients with cystic fibrosis will die before their 30th birthday. The cloning of the gene, which codes for a membrane protein designated the cystic fibrosis transmembrane conductance regulator, can thus be viewed in light of the need to achieve faster development of a treatment for the basic defect and, eventually, of a cure through drug therapy or gene therapy. Beyond the scope of CF research and treatment, the cloning of the CF gene also focuses attention on the international effort to map and sequence the human genome. We shall consider each of these aspects of the story. But since part of the story necessarily involves the Dr. Collins is Chief, Division of Medical Genetics, Departments of Internal Medicine and Human Genetics, University of Michigan Medical School, and Associate Investigator, Howard Hughes Medical Institute, Ann Arbor. Dr. Riordan is Professor of Biochemistry and Dr. Tsui is Professor of Medical Genetics, University of Toronto Faculty of Medicine. At the Hospital for Sick Children Research Institute, Toronto, Dr. Riordan is Senior Scientist, Department of Biochemistry, and Dr. Tsui is Senior Research Scientist, Department of Genetics.
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strategy by which this gene was isolated, we begin with a summary of techniques successfully used in the search.
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Strategy for Finding tbeCFGene Among the hundreds of human genes that have now been cloned, the CF gene was one of the more elusive. It was located by a process that has been called reverse genetics. Positional cloning may be a better description, since this process is not really genetics in reverse, just genetics. Whatever it is called, the search for the CF gene required a strategy that could proceed in the absence of any knowledge of the structure or function of the protein. Standard biochemical studies of genetic disorders proceed by isolation of the pertinent protein and its subsequent use to clone the gene. This straightforward approach works nicely. Unfortunately, it is not always possible, for a variety of reasons, such as lack of a functional assay. scarcity of the protein, and the complexity of protein interactions in the cell: sometimes, one cannot even identifY the type of cells affected. Cystic fibrosis studies were complicated by several of these problems. Studies over the past 10 years have revealed that cystic fibrosis results in abnormal ion transport across the apical membrane of epithelial cells of several tissues, including those of the respiratocy and gastrointestinal tract and sweat glands. But the biochemistty of the transport defect has remained murky, and the character of the defective protein was a mystecy. At a number of research laboratories, investigators decided to go directly to the genome to put their fingers on the CF gene be48
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fore the CF protein was in hand. Some of the techniques needed were already available in the early 1980s, when the hunt began: others would soon be developed. Nevertheless, the search for the CF gene turned out to be more difficult than had been anticipated. Simultaneous with the CF search, many investigators were using similar strategies to locate genes responsible for other single-gene disorders. Before the CF gene was cloned in 1989, positional cloning had already revealed the genes responsible for Duchenne muscular dystrophy, chronic granulomatous disease, and retinoblastoma. These successes depended on rare patients with gross chromosomal rearrangements involving the sought-after gene. Once investigators were able to piece together maps directing them to the right stretch of DNA. the mutations were large enough to stand out like signs along a highway, advertising the location of the defective gene. Unfortunately, the defect in the CF gene did not turn out to be well advertised. Although the gene itself is large (a 250-kilobase stretch of DNA on the long arm of chromosome 7), the defective segment that might have served as a signpost is small: A single trinucleotide codon is deleted in the common CF mutation. This deletion results in loss of one of the 1,480 amino acids that compose the normal protein. Even at this writing, no gross rearrangements of the gene have been found. In fact, the elusive gene was located through genetic linkage analysis, physical mapping, and molecular cloning. The first step in positional cloning is linking the gene to a particular chromosome. It is fairly easy to establish which
chromosome contains the gene for diseases that are sex-linked or characterized by observable cytogenetic abnormalities. For cystic fibrosis and other diseases without those markers, the gene must be mapped to its chromosome by genetic linkage analysis. Such analysis relies on two factors-one a product of human ingenuity and the other an important biologic phenomenon. The former is the revolution in molecular biology that has enabled investigators to clone "marker" segments of DNA More than 2,000 such segments of human DNA have been cloned and mapped to specific locations on specific chromosomes. A cloned marker is useful for genetic linkage analysis when it contains a polymorphism (a sequence that can vary from individual to individual). When a polymorphism (which can be as small as one base pair) creates or eliminates a recognition site for a restriction endonuclease, it is called a restriction fragment length polymorphism (RFLP). Different sequences of a particular marker represent alleles. The important biologic phenomenon is the recombination of genetic material that occurs during meiosis. Before chromosome pairs are separated and sealed in haploid sperm or egg cells, the parental chromosome pairs come together to exchange homologous sections of DNA (Figure 1). This recombination of DNA does not change the order of genes, but it may create new combinations of alleles. Linkage analysis is predicated on the observation that genes tend to be inherited together when located close to one another on the chromosome. These tightly "linked" segments will not be separated vety often
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in meiosis, simply because there is not much opportunity for recombination between them. Linkage between two marker segments or between a marker segment and a gene can be statistically analyzed. Segments far apart on a chromosome or on separate chromosomes are unlinked; they are inherited together 50% of the time. By definition, the distance between two DNA segments that recombine in 1% of progeny is 1 centimorgan (eM). It has been estimated that the entire human genome is 3,300 eM long. Since that length is roughly 3 billion (3x 10 9 ) base pairs, 1 eM is about 1 million base pairs. The exact physical length of a centimorgan varies, because recombination is not entirely random; some regions of the genome contain "hot spots" for recombination, and others relatively infrequently recombine. The first positive linkage results with cystic fibrosis were obtained in 1985, when Hans Eiberg and colleagues at the University Institute of Medical Genetics in Copenhagen reported linkage with the polymorphic enzyme paraoxonase. Unfortunately, the location of Figure 1. Recombination is the basis for linkage analysis, by which gene positions on chromosomes are mapped. During meiosis, DNA replicates and each chromosome becomes a pair of sister chromatids. Homologous chromosomes then become aligned (top). In the illustration, one chromosome carries alleles A and B, the other alleles a and b. The genetic material will be rearranged if there are crossovers between nonsister chromatids. If two genes are far apart (left sequence), there is a high probability that crossover will sever their "linkage," so that Ab and aB haplotypes appear among the resulting gametes. If the two genes are close together (right sequence), they are likely to remain in tandem, preserving the AB and ab linkages.
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the gene for that protein was sttll unknown. That same year, one of us (L.-C.T.), together with Manuel Buchwald in Toronto and Helen Donis-Ketler then in Boston, reported a linkage with a cloned segment of DNA. which established the CF gene's location on the long arm of chromosome 7. Other tightly linked polymorphtsms were quickly found within 1 to 2 eM of the CF gene: the met gene by Raymond White's laboratory at the University of Utah and J3.11 by Robert Wtlltamson's laboratory at St. Mary's Hospital in London. The tightly linked markers directed everyone's attention to a section of DNA 1 to 5 eM long. The exact position of the CF gene with respect to met and J3.11 was not entirely clear for more than two years. but the CF gene was eventually shown through a careful collaborative effort to lie between these two markers. This interval represents roughly one twentieth of 1% of the human genome and is about the best resolution obtainable from genetic linkage analysts, given the finite number of affected fam1ltes and the improbability of recombination as the area of interest becomes smaller. There sttll were up to 5 million base pairs left to search, and the next step was working directly with DNA to develop a physical map. This can be done in several ways. Cytogenetic mapping, using light microscopy to visualize chromosomal banding patterns, produces maps whose resolution is 5 million to 10 mtllton nucleotides. Standard molecular genetic restriction maps have much finer resolution, providing markers every 10 to 20 kb (1 kb = 1.000 base pairs). Restriction maps are made by cutting DNA with restriction endonucleases and 50
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separating the fragments according to size. The sizing of restriction fragments is done by standard agarose gel electrophoresis, for which the resolution breaks down above about 30kb. Two developments have made it possible to create physical maps whose resolution falls between standard restriction-site maps and cytogenetic maps. One was the discovery of restriction enzymes that recognize relatively rare sequences in the human genome, therefore producing larger fragments. The other was the development of pulsed-field gel electrophoresis (PFGE), which can resolve longer DNA segments. With these tools, it is possible to make maps of regions as large as 5 mtllton base pairs. By 1988, PFGE-generated physical maps of the CF region of chromosome 7 (Figure 2) had confirmed the genetic linkage analysts: The two markers that bracketed the CF gene according to genetic linkage analysts were in fact about 1,600 kb apart-a distance that matched the 1- to 5-cM figure. The PFGE map not only was necessary for determining the size of the region to be searched but also was helpful because it could rapidly establish the location of newly isolated clones that would be needed to reach the CF gene.
Cloning The maps provide starting points from which to move along the genome in search of the target gene. A variety of molecular cloning techniques are now possible. We used three to find the CF gene: saturation cloning, chromosome walking, and chromosome jumping. In an effort to find a starting point closer to the gene, one of
us (L.-C.T.) applied the bruteforce tactic of saturation cloning. By isolating and mapping a great many random clones from chromosome 7, a marker even closer to the target should be identified. By mid-1987, 258 random clones from chromosome 7 had been isolated. Fiftythree mapped to the CF region, and linkage analysts showed that two were closer to the CF gene than any previous cloned segment (Figure 2). Surprisingly, the two random clones, D7S122 and D7S340, were separated from each other by only 10 kb. Unfortunately. they appeared to be several hundred kilobases away from the CF gene. Chromosome walking involves repeated cloning of overlapping DNA segments. The largest conventional cloning vectors are cosmtds, which yield about 40 kb of DNA with each clone. Chromosome walking is a slow, laborious way to move along a chromosome. The distance covered is rarely more than 100 to 200 kb, beyond which an unclonable stretch of DNA often brings the walk to a dead end. To travel greater distances along the genome, one of us (F.S.C.) developed the strategy of chromosome jumping. To jump along a chromosome, DNA was cut into fragments as long as a hundred thousand base pairs. These fragments are too long to be easily walked, but they can be bent into circles. When we cloned the segment where the circle is joined, we were actually cloning sequences that, although brought together in the laboratory, naturally occur far apart. Jumping along the chromosome provided two advantages: We could move more quickly, and we could skip over unclonable segments. With these techniques, and
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with the consolidation of the Ann Arbor and Toronto laboratories into an intensive collaborative effort, we had moved, by the end of 1988, from the linked markers into the area where we thought the gene must be. The question then became one of recognition: how to know whether the material cloned actually contained a gene and, if so, how to determine whether it was the CF gene. Genes can be identified by several features. Many genes have been conserved through evolution and therefore contain DNA sequences that will crosshybridize between species. This distinguishes between such DNA sequences and the 85% of the human genome that does not seem to be involved in coding for proteins. A second clue is the identification of a stretch of DNA. 500 to 2,000 base pairs long, that is rich in the nonmethylated nucleotides cytosine and guanosine. In vertebrates, a CG "island" such as this often marks the beginning of a gene. Third, a DNA segment can also be identified as part of a gene by screening messenger RNA (mRNA) or complementary DNA (eDNA) libraries for evidence of transcription in affected tissues. The presence or absence of one of these signs is not conclusive; often, all must be used to identify a segment as part of a gene. Even after walking and jumping our way to the CF gene, we were not at all sure that we had the right gene. as it was expected that there would be other genes in the neighborhood. By December 1988, we had cloned what turned out to be a piece of the gene but lacked evidence that it was transcribed. As it happens, that evidence was elusive for two reasons: The transcript (mRNA) of the gene re-
Figure 2. Preliminary search for the gene responsible for cystic fibrosis relied on linkage analysis, which placed the CF gene on the long arm of chromosome 7, in close linkage to two identifiable markers, the met oncogene and an anonymous probe designated J3.11. Investigation with a variety of molecular biologic techniques established that these markers bracket a length of more than 1,500 kilobases (blue). Two closely spaced markers identified by Lap-Chee Tsui (075122 and 075340) were then shown to be even more tightly linked to the CF gene.
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, Walking and Jumping
MSOB /Exon
f (( Tsui's, Probes
(( [ (((([f [[[ ( (( (((( {}
'' I '.
t
t
t
kb
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Figure 3. The CF gene begins more than 250 kilobases away from Tsui's probes. The search proceeded by "walking" (the cloning and sequencing of overlapping segments of DNA along chromosome 7), with numerous "jumps" over unclonable or uninteresting regions. Three times, a nucleotide sequence suggesting the start of a gene was discovered (arrows); the third of these was the
sponsible for CF, which has been named the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is rather low in abundance, and the fragment studied actually contained only the first of the gene's exons. That exon is comparatively small, containingjust 113 base pairs. The rare message and short sequence combined to scuttle initial attempts to find our segment represented in mRNA. The segment was. however, conserved across species and had a CG island, so we continued to look for transcription. Finally. early in 1989, one of us (J.R.R.). whose laboratory had earlier joined this collaborative effort, obtained a eDNA match in a sweat gland library. This eDNA library, as well as that subsequently used to detect the major mutation, was constructed by using mRNA from cultured sweat gland epithelial cells that retained the anion permeability pathway defective in cystic fibrosis. The initial eDNA clone contained exon 1 and eight more exons. Once we knew that we had a gene. we isolated overlapping eDNA clones to piece together the 52
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start of the CF gene. The gene itself is remarkably longabout 250 kilobases-but exons (sequences translated into protein) compose only about 2.5% of its length. In about 70% of CF patients, three base pairs are missing from exon 10 (red dot), with a resulting phenylalanine deletion from amino acid position 508 (designated t:.F508) in the protein specified by the gene.
whole gene, which was found to span a surprisingly large 250kb region (genes are usually on the order of 20 kb). Most of this gene was represented by intrans. but the exons coded for a 6.5-kb mRNA. which predicted a protein of 1.480 amino acids. We had a large and interesting gene. Was it the CF gene? In the absence of information on the protein or a functional assay. we could prove the correctness of the candidate only by finding a mutation in CF patients that never occurs in normal chromosomes. But how can we identifY a chromosome that is definitively normal? One cannot assume that people who do not have cystic fibrosis have normal chromosomes, because 5% of unaffected whites do carry one CF gene. Oddly enough. the place to look for an assuredly normal chromosome is in the parents of children with cystic fibrosis. Each biologic parent of a child with CF must have one CF gene and one normal gene. The researchers in Toronto found a three-base pair deletion in a eDNA sample from a library constructed with RNA isolated from epithelial cells cui-
tured from a CF patient's sweat glands. This deletion leads to the loss of a single amino acid (phenylalanine) at position 508 of CFTR (usually referred to as b.F508). This initially seemed likely to be a polymorphism. since there were reasons to expect the CF mutation to be something more gross: a nonsense mutation that would stop translation altogether or a nonconservative missense mutation that would substitute one amino acid for another. But by June 1989, more than 200 CF parents had been studied who had the common mutation on one copy of chromosome 7, and none of them had the same mutation on the other copy (their normal chromosome). At that point. we were confident that we had the CF gene (Figure 3). The data on normal chromosomes now number in the thousands and support our earlier conclusion. Not all mutations responsible for CF are due to b.F508, however. About 25% to 30% of CF chromosomes in whites do not carry b.F508, which indicates that other mutations in CFTR can also be responsible. Intensive efforts from a large consor-
tium of laboratories around the world have so far identified about 50 additional CF mutations, most of which are uncommon in the population.
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Carrier Screening Since one in 20 white North Americans carries the CF gene, effective carrier screening could be clinically important. Within months of the gene's cloning. DNA diagnostic companies and a number of research laboratories were already offering screening to families with a history of cystic fibrosis, where the information can often be of considerable value to the couple at risk (Figure 4). Unfortunately, the tests currently available detect just 70% to 75% of carriers (those with AF508), a level of sensitivity that can cause problems. Despite the potential benefits of carrier screening, careful consideration indicates that the medical profession should not rush into general screening. With testing now available, half of the couples at risk of having a child with cystic fibrosis would be missed. Nor could anyone be assured that he or she does not carry the gene, since 25% to 30% of carriers would be missed by AF508 testing. Because of these uncertainties, an NIH workshop of geneticists, ethicists. and public health officials concluded in March 1990 that across-theboard carrier screening for cystic fibrosis should not yet be considered an applicable standard of medical care. This caution grows in part out of a concern for couples in which one partner tests positive and the other negative. Without screening, the standard risk of a white child's being affected in the absence of a family
history of cystic fibrosis is only one in 2,500. If the parents-tobe are screened for AF508 and one is positive and one negative, suddenly the risk estimated for CF increases to one in 400. Therefore, for couples such as this, who will not be uncommon, the result of screening is likely to be a heightened level of anxiety. In addition, the frequency of the disease and the types of mutations vary according to racial and ethnic backgrounds. Therefore, different laboratory procedures and counseling modifications will be required for different populations. Another major consideration is that the public will need to be better informed about genetics and screening before it can be expected to understand the limitations and uses of an imperfect screening test. Any screening program will have to include a major educational arm, one that can help people make an informed decision about whether they want the test. and offer counseling about the result. For couples who do have a family history of cystic fibrosis on one side or the other, carrier screening is already appropriate. For such couples, the currently available test is likely to give useful information about their odds of having an affected child. Such couples can and should be offered genetic counseling by their primacy physician or through referral to a genetic center. The best time for prospective screening of couples is bEfore conception, when more options are available. For some couples, prenatal diagnosis followed by abortion of an affected fetus is not an acceptable option. Indeed, to what extent that option will be chosen even by those
Figure 4. CF carrier screening now identifies only the most common defect, ~F508, but is useful for couples with a CF family history. A child of two carriers has a 1/4 chance of being affected; an unaffected child has a 2/3 chance of being a carrier. Before testing, the mate of a carrier (assuming Northern European background) has the 1/25 probability of the general population. The probability that both would transmit an abnormality is 1/4, and the likelihood of disease is 1/150 (top). If both parents test positive (middle), the likelihood increases to 1/4. If only the person with a family history tests positive (bottom), it falls but not to zero, because the other partner has a 1/100 chance of carrying an unidentifiable CF defect.
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philosophically comfortable with abortion is unclear. Unlike Tay-Sachs disease and other diseases for which screening was very much welcomed by those at risk, cystic fibrosis is compatible with many years of good-quality life. Median survival now extends to age 26. Whether one talks of prenatal or carrier screening, negative or positive family histories, or tests that are 80%, 90%, or 95% accurate, it is probable that some type of CF screening will eventually be offered. It is equally clear that geneticists, ethicists, and public health officials should take a hard look at the issue of screening. As a negative example, the experience with sickle cell screening should be recalled. Some of those screened for sickle cell trait were not well informed about why the screening was being done and what the results meant. Insurance companies were inappropriately involved and often confused about the difference between being a carrier and having the disease. Such a tragic situation must not be allowed to recur. Pilot projects on well-defined populations, with careful assessment of the outcome, are essential to a successful CF screening program.
1be Basic Defect Although identification of the CF gene did not instantly change clinical medicine, it did immediately focus the research effort. Less than two months after announcement of the gene's cloning, more than 70 laboratories around the world joined forces to explore the gene in a coordinated manner. This unprecedented consortium focuses on the mutations that cause cystic fibrosis. As previously noted, one identified 54
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mutation-the single deleted codon at position 508 (.6.F508) with a consequent loss of a phenylalanine-is found in about 70% of CF chromosomes. Members of the consortium share information about other potentially deleterious mutations well before submission for publication-a remarkable arrangement that prevents pointless duplication of effort and hastens progress toward a better understanding of the disease.
1be CFIR Protein With the gene cloned, we were able to predict the amino acid sequence of the long-elusive CF protein: the cystic fibrosis transmembrane conductance regulator. Of course, the sequence did not tell us everything about what CFTR is doing in the cell. But as long as the gene can, as it does, predict the primary structure of the protein, it raises to a virtual certainty the likelihood that investigators will eventually find the molecular answer to the question, what is cystic fibrosis? Initially, the agenda calls for exploring CITR's role in regulating transport of chloride ions out of epithelial cells. Is CITR directly involved in ion conductance, or does it indirectly regulate that process? To find out. we need a functional assay. We must study CITR in its normal setting (a respiratory epithelial cell or a sweat gland cell), as well as in cells not normally involved with chloride transport. An important step will be to put the protein into cells that do not normally transport chloride ions. Xenopus oocytes are a promising vehicle, since both endogenous and exogenous membrane channels have been well studied in them. Experi-
ments with Xenopus oocytes could help clear up the question of how CITR is involved in the regulation of impermeability. It will also be important to put the normal CITR gene into CF cells to see if the defective cells begin to regulate transport of chloride properly. If this works, it will provide a system for testing the many mutations found in the large CF gene. The goal would be to distinguish between serious defects and harmless polymorphisms. Not long after the gene was cloned, a number of laboratories began trying to make an antibody against CITR. one that could serve as a probe to pinpoint the protein's location in the cell. By examining the CF gene, it is clear that CITR has hydrophobic domains that almost certainly indicate that the protein will be found in cell membranes (Figure 5). This is, of course, no surprise, but with a fluorescently labeled antibody, investigators will be able to locate CITR conclusively. Another important item on the agenda is development of an animal model for cystic fibrosis. With the gene cloned, it should be possible to create such a model and use it to study the biochemistry of CF and to test potential CF therapies. The human CF gene is very similar in sequence to that of the murine counterpart. Using new site-specific recombination technologies, several groups are attempting to develop a mouse strain that carries the f1F508 CF mutation. If such mice prove to have a disease resembling cystic fibrosis, this could provide a valuable model.
Drug Treatment Drug treatment for CF patients is currently limited to
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pancreatic enzyme replacement and to antibiotic therapy for the bacterial infections associated with mucous accretions in the lungs. Except for a recent. promising small trial with aerosolized amiloride, a drug that alters sodium transport. no drugs have been found to positively influence ion transport and prevent the life-threaten-
ing accumulation of mucus. With the CF gene cloned, however, research on treatment can proceed along several lines, some of them even before we have a biochemical understanding of the basic defect. The most straightforward approach would be to produce large amounts of the normal CFTR protein and deliver it to
Cytosol
~
R Domain
Figure 5. The protein encoded by the CF gene has been termed cystic fibrosis transmembrane conductance regulator (CFTR). Its 12 hydrophobic domains appear to anchor the molecule in the plasma membrane, with little of its structure exposed at the cell surface. The portion inside the cell is notable for a large globular region called the R domain and for two smaller regions, nucleotide
affected lung tissues. Since cystic fibrosis is a recessive disease and carriers have 50% abnormal protein but are healthy, we know that the defective protein need not be eliminated. All that is needed is enough normal protein, and in some recessive diseases, as little as 5% normal protein is enough for normal cell function.
ATP Binding 'Domains
~F508 / Mutation
binding folds (NBFs), that are probably capable of binding ATP. The ~F508 defect found in most CF patients (red dot) is in the region of one of the NBFs. With its transmembrane spans and NBFs, the structure of CFTR closely resembles that seen in a family of transport proteins that includes P-glycoprotein, an ATP-binding pump that functions physiologically to rid cells of toxins.
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Although cystic fibrosis is a multiorgan disease, the lungs are the critical target for treatment. It might be possible to aerosolize a suspension of CFfR protein, perhaps inserted into a lipid bilayer that is capable of fusing spontaneously with the cell membrane. How to get the protein into membranes of the right cells (ciliated respiratory epithelial cells) is, however, a major challenge. A trial-and-error search for drugs that promote chloride transport can also proceed in advance of understanding the basic defect. To test the tens of thousands of compounds in stock, a drug company would need an ample supply of the CF version of CFfR and a simple assay for chloride transportperhaps even in an artificial membrane. If this sounds lowtech and inefficient, it is pertinent to recall that most drugs were discovered through trial and error. Rational drug design is still possible, provided progress in understanding the basic defect can be made. If a CF model can be developed and the chloride transport pathway can be dissected, it may be possible to determine what agonists or antagonists promote chloride transport.
Gene 1berapy Gene therapy could be the ultimate intervention. If the normal CFTR gene can be inserted into the right cells, the potential for cure, not just amelioration, could be realized. Having said this, it must be cautioned that gene therapy for cystic fibrosis is unlikely to be available in the near future. But CF gene therapy is a plausible possibility. In many diseases that might benefit from such therapy, gene 56
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delivery is complicated by the affected tissue's inaccessibility. In CF, the airways are relatively accessible, essentially noninvasively. In fact, it has already been shown that retroviruses penetrate rodent respiratory epithelial cells with reasonable efficiency. Retroviruses have often been used to deliver genes because they are efficient at getting into cells and integrating their DNA into the cells' DNA if the cells are dividing. Other viruses may tum out to be better delivery systems. Adenoviruses readily infect the respiratory tract, and much is known about their biology. They have already been used for gene transfer in tissue culture. We know less about respiratory syncytial viruses, but they may also be vector candidates. Conceivably, the best vector may be something like a synthetic lipid vesicle, rather than a virus. Other crucial issues to be addressed before this approach can be successful are the identity of the ideal target cells, the kinetics of cell division, how much protein to make inside the cells, and what proportion of cells need to be transfected in order to improve the patient's health. These questions are beginning to be explored. Much of the preliminary work must be done in animal models. While gene therapy offers great promises, it also poses problems. Clearly, research needs to continue on all fronts simultaneously-gene therapy, drug treatment, and studies of the basic defect.
1be Human Genome Project The success of positional cloning in locating the CF gene confirms something important about the project to identify
human disease genes by mapping and sequencing the human genome: It works. It can be done. A skeptic might point out that the search for this one gene involved tremendous labor and a huge amount of money. What about mapping and sequencing no less than the entire human genome? A large and well-financed collection of laboratories spent more than five years searching for the CF gene, along the way cloning and mapping roughly one part in 6,000 of the human genomeand sequencing only a small fraction of what was cloned. If the entire human genome were to be mapped and sequenced at the same rate, it would take a long, long time. Were the technology for mapping and sequencing the genome static, the human genome project would be in trouble. But the technology is anything but static. Progress has been steady. The yeast artificial chromosome system is one powerful new technique not available during the search for the CF gene. It allows cloning of a region 400 or 500 kb long. It took us almost two years to cross the 300-kb interval between CF markers and the CF gene. Had the yeast artificial chromosome technology been available, the walking and jumping process could have been accelerated. Another new technique, used successfully by one of us (F.S.C.) in the recent identification of the neurofibromatosis gene, might allow the identification of all transcripts in a given region in one experiment instead of many. In this approach, a yeast artificial chromosome is used directly to probe a eDNA library: if successful, all of the transcripts in a 200- to 500-kb region can be
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identified in one experiment rather than via the painstaking, fragment-by-fragment search that was carried out for cystic fibrosis. Mapping and sequencing the human genome's entire 3 billion base pairs is a huge undertaking. Assuming one character per nucleotide, an individual human's haploid genome would fill 13 sets of the Encyclopaedia Brttanntca. Not 13 volumes-13 complete sets. Of course, the project is bigger than any one person's genome. The goal is to be able to find and sequence some of the great variety in the human genomepolymorphisms and mutations alike. And to make sense of this information, it will be necessary concurrently to build maps and sequences of other species. It seems clear that a focused international effort will be more efficient than an ad hoc approach to gathering, storing, and learning to use such a large volume of important data. Some have questioned whether having a complete map and sequence will expedite the process of finding disease genes. One might argue that the genes involved in important diseases, such as cystic fibrosis and Huntington's disease, are going to be found anyway. Many will no doubt be located before the project is advanced enough to aid the search. But there are many harder problems-schizophrenia and Alzheimer's disease. for instance-in which it has been very difficult to locate the responsible gene. In truth, it is hard to imagine current strategies pinpointing the gene for Alzheimer's disease, which in some families seems to be hidden somewhere in a 5 millionto 10 million-base pair section of DNA on chromosome 21 that houses perhaps 100 genes.
The problem is further complicated by the evidence that Alzheimer genes exist on other chromosomes in other families. With a complete map and sequence of the 1 million to 10 million base pairs in the regions of interest. computer analysis could potentially identify most of the coding regions that indicate the presence of genes. One could then look at the DNA sequences of the putative genes to see which code for proteins that look homologous to other proteins in the central nervous system. With the normal sequences of CNS genes already identified as part of the genome project, the relevant DNA of Alzheimer's patients could be much more readily screened for mutations. In diseases that involve several genes, the mapping and precise localization of responsible loci are certain to be more difficult even than problems postulated for Alzheimer's disease. Thus, when the genetic basis of disorders such as hypertension, coronary artery disease,
and breast cancer is pursued, there is not likely to be much success until considerable map information is available. Another crucial need of this effort is to collect DNA from many families with the diseases to be studied. A detailed map and complete sequence of normal, healthy genes will not, by themselves, reveal very much about diseases. The genetic analysis that points the way to disease genes depends on a large number of DNA samples from affected families. In summary, the search for the CF gene has taught us much about the positional cloning strategy, and it is likely that many other genes for human disorders will be found in this fashion, especially with the advances expected from the human genome project. The current challenge in CF research is to use the newly found knowledge about CITR to deduce the basic biochemical defect, and to develop an effective treatment for those with the disease. D
Selected Reading Rommens JM et al: Identification of the cystic fibrosis gene: Chromosome walking and jumping. Science 245: 1059. 1989 Riordan JR et al: Identification of the cystic fibrosis gene: Cloning and characterization of complementaiy DNA Science 245: 1066. 1989 Kerem B et al: Identification of the cystic fibrosis gene: Genetic analysis. Science 245:1073. 1989 Iannuzzi MC. Collins FS: Update: Reverse genetics and cystic fibrosis. Am J Respir Cell Mol Bioi 2:309. 1990 Lemna WK et al: Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis. N Engl J Med 322:291. 1990 Statement from the NIH Workshop on Population Screening for the Cystic Fibrosis Gene. N Engl J Med 323:70. 1990 Orkin S: Reverse genetics and human disease. Cell 4 7:845, 1986 WhiteR, Lalouel J-M: Chromosome mapping with DNA markers. Sci Am 258:40, Februacy 1988 McKusick VA: Mapping and sequencing the human genome. N Engl J Med 320:910, 1989 Watson JD: The human genome project: Past, present, and future. Science 248:44, 1990
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ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
The Cystic Fibrosis Gene: Isolation and Significance Francis S. Collins, John R. Riordan & Lap-Chee Tsui To cite this article: Francis S. Collins, John R. Riordan & Lap-Chee Tsui (1990) The Cystic Fibrosis Gene: Isolation and Significance, Hospital Practice, 25:10, 47-57, DOI: 10.1080/21548331.1990.11704019 To link to this article: http://dx.doi.org/10.1080/21548331.1990.11704019
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The Cystic Fibrosis Gene: Isolation and Significance and LAP-CHEE University of Michigan and University qf Toronto
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FRANCIS S. COLLINS, JOHN R RIORDAN,
TSUI
The identification and cloning of the CF gene, which codes for a membrane protein that appears to regulate transmembrane ion transport, may lead to an understanding of the basic defect in the disease and to more effective treatment. More broadly, the cloning of the CF gene provides a fast start in the international effort to clone and map the entire human genome.
The search for the cystic fibrosis gene was a long and arduous process that required considerable time, energy, and money. Two research groups in Ann Arbor, Mich., and Toronto, working in close collaboration, finally identified the gene in September 1989-almost a decade after the search began. Almost as soon as the gene was in hand, improvements in prenatal diagnosis and carrier screening became possible. The therapeutic benefits that may follow from cloning the gene, however, are still probably some years away. The expectation is that they will arise from a better understanding of the basic biologic problem and perhaps involve gene therapy. The identification of the CF gene represented a solution to a challenging problem, one that has given additional momentum to the efforts to map and sequence the human genome. That genome is usually described as a library of 3 billion characters or as a blueprint for building human beings. The metaphors imply that if one gains access to the library and knows how to solve the code, one can read the approximately 100,000 human genes like an owner's manual. The manual is not very "user-friendly,'' however. Genes (DNA} code for RNA. which codes for proteins. Where those proteins go, what they do, how they interact with other proteins or fail to interact are exceedingly difficult biochemical questions. The answers may be written in the genes but are not directly interpretable with current knowledge. With this perspective kept in mind, we can now proceed to review the work involved in identification of the CF gene.
Cystic fibrosis affects an estimated 30,000 young Americans, and an additional 2,000 babies are born with the disease every year. It is the most common severe autosomal recessive disease in whites. Antibiotic therapy of secondary bacterial infections, improved chest physical therapy. and better nutrition have increased life expectancy in CF patients. but little progress has been made in treating the basic defect. The disturbing prognosis remains: Without dramatic clinical advances, most patients with cystic fibrosis will die before their 30th birthday. The cloning of the gene, which codes for a membrane protein designated the cystic fibrosis transmembrane conductance regulator, can thus be viewed in light of the need to achieve faster development of a treatment for the basic defect and, eventually, of a cure through drug therapy or gene therapy. Beyond the scope of CF research and treatment, the cloning of the CF gene also focuses attention on the international effort to map and sequence the human genome. We shall consider each of these aspects of the story. But since part of the story necessarily involves the Dr. Collins is Chief, Division of Medical Genetics, Departments of Internal Medicine and Human Genetics, University of Michigan Medical School, and Associate Investigator, Howard Hughes Medical Institute, Ann Arbor. Dr. Riordan is Professor of Biochemistry and Dr. Tsui is Professor of Medical Genetics, University of Toronto Faculty of Medicine. At the Hospital for Sick Children Research Institute, Toronto, Dr. Riordan is Senior Scientist, Department of Biochemistry, and Dr. Tsui is Senior Research Scientist, Department of Genetics.
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strategy by which this gene was isolated, we begin with a summary of techniques successfully used in the search.
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Strategy for Finding tbeCFGene Among the hundreds of human genes that have now been cloned, the CF gene was one of the more elusive. It was located by a process that has been called reverse genetics. Positional cloning may be a better description, since this process is not really genetics in reverse, just genetics. Whatever it is called, the search for the CF gene required a strategy that could proceed in the absence of any knowledge of the structure or function of the protein. Standard biochemical studies of genetic disorders proceed by isolation of the pertinent protein and its subsequent use to clone the gene. This straightforward approach works nicely. Unfortunately, it is not always possible, for a variety of reasons, such as lack of a functional assay. scarcity of the protein, and the complexity of protein interactions in the cell: sometimes, one cannot even identifY the type of cells affected. Cystic fibrosis studies were complicated by several of these problems. Studies over the past 10 years have revealed that cystic fibrosis results in abnormal ion transport across the apical membrane of epithelial cells of several tissues, including those of the respiratocy and gastrointestinal tract and sweat glands. But the biochemistty of the transport defect has remained murky, and the character of the defective protein was a mystecy. At a number of research laboratories, investigators decided to go directly to the genome to put their fingers on the CF gene be48
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fore the CF protein was in hand. Some of the techniques needed were already available in the early 1980s, when the hunt began: others would soon be developed. Nevertheless, the search for the CF gene turned out to be more difficult than had been anticipated. Simultaneous with the CF search, many investigators were using similar strategies to locate genes responsible for other single-gene disorders. Before the CF gene was cloned in 1989, positional cloning had already revealed the genes responsible for Duchenne muscular dystrophy, chronic granulomatous disease, and retinoblastoma. These successes depended on rare patients with gross chromosomal rearrangements involving the sought-after gene. Once investigators were able to piece together maps directing them to the right stretch of DNA. the mutations were large enough to stand out like signs along a highway, advertising the location of the defective gene. Unfortunately, the defect in the CF gene did not turn out to be well advertised. Although the gene itself is large (a 250-kilobase stretch of DNA on the long arm of chromosome 7), the defective segment that might have served as a signpost is small: A single trinucleotide codon is deleted in the common CF mutation. This deletion results in loss of one of the 1,480 amino acids that compose the normal protein. Even at this writing, no gross rearrangements of the gene have been found. In fact, the elusive gene was located through genetic linkage analysis, physical mapping, and molecular cloning. The first step in positional cloning is linking the gene to a particular chromosome. It is fairly easy to establish which
chromosome contains the gene for diseases that are sex-linked or characterized by observable cytogenetic abnormalities. For cystic fibrosis and other diseases without those markers, the gene must be mapped to its chromosome by genetic linkage analysis. Such analysis relies on two factors-one a product of human ingenuity and the other an important biologic phenomenon. The former is the revolution in molecular biology that has enabled investigators to clone "marker" segments of DNA More than 2,000 such segments of human DNA have been cloned and mapped to specific locations on specific chromosomes. A cloned marker is useful for genetic linkage analysis when it contains a polymorphism (a sequence that can vary from individual to individual). When a polymorphism (which can be as small as one base pair) creates or eliminates a recognition site for a restriction endonuclease, it is called a restriction fragment length polymorphism (RFLP). Different sequences of a particular marker represent alleles. The important biologic phenomenon is the recombination of genetic material that occurs during meiosis. Before chromosome pairs are separated and sealed in haploid sperm or egg cells, the parental chromosome pairs come together to exchange homologous sections of DNA (Figure 1). This recombination of DNA does not change the order of genes, but it may create new combinations of alleles. Linkage analysis is predicated on the observation that genes tend to be inherited together when located close to one another on the chromosome. These tightly "linked" segments will not be separated vety often
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in meiosis, simply because there is not much opportunity for recombination between them. Linkage between two marker segments or between a marker segment and a gene can be statistically analyzed. Segments far apart on a chromosome or on separate chromosomes are unlinked; they are inherited together 50% of the time. By definition, the distance between two DNA segments that recombine in 1% of progeny is 1 centimorgan (eM). It has been estimated that the entire human genome is 3,300 eM long. Since that length is roughly 3 billion (3x 10 9 ) base pairs, 1 eM is about 1 million base pairs. The exact physical length of a centimorgan varies, because recombination is not entirely random; some regions of the genome contain "hot spots" for recombination, and others relatively infrequently recombine. The first positive linkage results with cystic fibrosis were obtained in 1985, when Hans Eiberg and colleagues at the University Institute of Medical Genetics in Copenhagen reported linkage with the polymorphic enzyme paraoxonase. Unfortunately, the location of Figure 1. Recombination is the basis for linkage analysis, by which gene positions on chromosomes are mapped. During meiosis, DNA replicates and each chromosome becomes a pair of sister chromatids. Homologous chromosomes then become aligned (top). In the illustration, one chromosome carries alleles A and B, the other alleles a and b. The genetic material will be rearranged if there are crossovers between nonsister chromatids. If two genes are far apart (left sequence), there is a high probability that crossover will sever their "linkage," so that Ab and aB haplotypes appear among the resulting gametes. If the two genes are close together (right sequence), they are likely to remain in tandem, preserving the AB and ab linkages.
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the gene for that protein was sttll unknown. That same year, one of us (L.-C.T.), together with Manuel Buchwald in Toronto and Helen Donis-Ketler then in Boston, reported a linkage with a cloned segment of DNA. which established the CF gene's location on the long arm of chromosome 7. Other tightly linked polymorphtsms were quickly found within 1 to 2 eM of the CF gene: the met gene by Raymond White's laboratory at the University of Utah and J3.11 by Robert Wtlltamson's laboratory at St. Mary's Hospital in London. The tightly linked markers directed everyone's attention to a section of DNA 1 to 5 eM long. The exact position of the CF gene with respect to met and J3.11 was not entirely clear for more than two years. but the CF gene was eventually shown through a careful collaborative effort to lie between these two markers. This interval represents roughly one twentieth of 1% of the human genome and is about the best resolution obtainable from genetic linkage analysts, given the finite number of affected fam1ltes and the improbability of recombination as the area of interest becomes smaller. There sttll were up to 5 million base pairs left to search, and the next step was working directly with DNA to develop a physical map. This can be done in several ways. Cytogenetic mapping, using light microscopy to visualize chromosomal banding patterns, produces maps whose resolution is 5 million to 10 mtllton nucleotides. Standard molecular genetic restriction maps have much finer resolution, providing markers every 10 to 20 kb (1 kb = 1.000 base pairs). Restriction maps are made by cutting DNA with restriction endonucleases and 50
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separating the fragments according to size. The sizing of restriction fragments is done by standard agarose gel electrophoresis, for which the resolution breaks down above about 30kb. Two developments have made it possible to create physical maps whose resolution falls between standard restriction-site maps and cytogenetic maps. One was the discovery of restriction enzymes that recognize relatively rare sequences in the human genome, therefore producing larger fragments. The other was the development of pulsed-field gel electrophoresis (PFGE), which can resolve longer DNA segments. With these tools, it is possible to make maps of regions as large as 5 mtllton base pairs. By 1988, PFGE-generated physical maps of the CF region of chromosome 7 (Figure 2) had confirmed the genetic linkage analysts: The two markers that bracketed the CF gene according to genetic linkage analysts were in fact about 1,600 kb apart-a distance that matched the 1- to 5-cM figure. The PFGE map not only was necessary for determining the size of the region to be searched but also was helpful because it could rapidly establish the location of newly isolated clones that would be needed to reach the CF gene.
Cloning The maps provide starting points from which to move along the genome in search of the target gene. A variety of molecular cloning techniques are now possible. We used three to find the CF gene: saturation cloning, chromosome walking, and chromosome jumping. In an effort to find a starting point closer to the gene, one of
us (L.-C.T.) applied the bruteforce tactic of saturation cloning. By isolating and mapping a great many random clones from chromosome 7, a marker even closer to the target should be identified. By mid-1987, 258 random clones from chromosome 7 had been isolated. Fiftythree mapped to the CF region, and linkage analysts showed that two were closer to the CF gene than any previous cloned segment (Figure 2). Surprisingly, the two random clones, D7S122 and D7S340, were separated from each other by only 10 kb. Unfortunately. they appeared to be several hundred kilobases away from the CF gene. Chromosome walking involves repeated cloning of overlapping DNA segments. The largest conventional cloning vectors are cosmtds, which yield about 40 kb of DNA with each clone. Chromosome walking is a slow, laborious way to move along a chromosome. The distance covered is rarely more than 100 to 200 kb, beyond which an unclonable stretch of DNA often brings the walk to a dead end. To travel greater distances along the genome, one of us (F.S.C.) developed the strategy of chromosome jumping. To jump along a chromosome, DNA was cut into fragments as long as a hundred thousand base pairs. These fragments are too long to be easily walked, but they can be bent into circles. When we cloned the segment where the circle is joined, we were actually cloning sequences that, although brought together in the laboratory, naturally occur far apart. Jumping along the chromosome provided two advantages: We could move more quickly, and we could skip over unclonable segments. With these techniques, and
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with the consolidation of the Ann Arbor and Toronto laboratories into an intensive collaborative effort, we had moved, by the end of 1988, from the linked markers into the area where we thought the gene must be. The question then became one of recognition: how to know whether the material cloned actually contained a gene and, if so, how to determine whether it was the CF gene. Genes can be identified by several features. Many genes have been conserved through evolution and therefore contain DNA sequences that will crosshybridize between species. This distinguishes between such DNA sequences and the 85% of the human genome that does not seem to be involved in coding for proteins. A second clue is the identification of a stretch of DNA. 500 to 2,000 base pairs long, that is rich in the nonmethylated nucleotides cytosine and guanosine. In vertebrates, a CG "island" such as this often marks the beginning of a gene. Third, a DNA segment can also be identified as part of a gene by screening messenger RNA (mRNA) or complementary DNA (eDNA) libraries for evidence of transcription in affected tissues. The presence or absence of one of these signs is not conclusive; often, all must be used to identify a segment as part of a gene. Even after walking and jumping our way to the CF gene, we were not at all sure that we had the right gene. as it was expected that there would be other genes in the neighborhood. By December 1988, we had cloned what turned out to be a piece of the gene but lacked evidence that it was transcribed. As it happens, that evidence was elusive for two reasons: The transcript (mRNA) of the gene re-
Figure 2. Preliminary search for the gene responsible for cystic fibrosis relied on linkage analysis, which placed the CF gene on the long arm of chromosome 7, in close linkage to two identifiable markers, the met oncogene and an anonymous probe designated J3.11. Investigation with a variety of molecular biologic techniques established that these markers bracket a length of more than 1,500 kilobases (blue). Two closely spaced markers identified by Lap-Chee Tsui (075122 and 075340) were then shown to be even more tightly linked to the CF gene.
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, Walking and Jumping
MSOB /Exon
f (( Tsui's, Probes
(( [ (((([f [[[ ( (( (((( {}
'' I '.
t
t
t
kb
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Figure 3. The CF gene begins more than 250 kilobases away from Tsui's probes. The search proceeded by "walking" (the cloning and sequencing of overlapping segments of DNA along chromosome 7), with numerous "jumps" over unclonable or uninteresting regions. Three times, a nucleotide sequence suggesting the start of a gene was discovered (arrows); the third of these was the
sponsible for CF, which has been named the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is rather low in abundance, and the fragment studied actually contained only the first of the gene's exons. That exon is comparatively small, containingjust 113 base pairs. The rare message and short sequence combined to scuttle initial attempts to find our segment represented in mRNA. The segment was. however, conserved across species and had a CG island, so we continued to look for transcription. Finally. early in 1989, one of us (J.R.R.). whose laboratory had earlier joined this collaborative effort, obtained a eDNA match in a sweat gland library. This eDNA library, as well as that subsequently used to detect the major mutation, was constructed by using mRNA from cultured sweat gland epithelial cells that retained the anion permeability pathway defective in cystic fibrosis. The initial eDNA clone contained exon 1 and eight more exons. Once we knew that we had a gene. we isolated overlapping eDNA clones to piece together the 52
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start of the CF gene. The gene itself is remarkably longabout 250 kilobases-but exons (sequences translated into protein) compose only about 2.5% of its length. In about 70% of CF patients, three base pairs are missing from exon 10 (red dot), with a resulting phenylalanine deletion from amino acid position 508 (designated t:.F508) in the protein specified by the gene.
whole gene, which was found to span a surprisingly large 250kb region (genes are usually on the order of 20 kb). Most of this gene was represented by intrans. but the exons coded for a 6.5-kb mRNA. which predicted a protein of 1.480 amino acids. We had a large and interesting gene. Was it the CF gene? In the absence of information on the protein or a functional assay. we could prove the correctness of the candidate only by finding a mutation in CF patients that never occurs in normal chromosomes. But how can we identifY a chromosome that is definitively normal? One cannot assume that people who do not have cystic fibrosis have normal chromosomes, because 5% of unaffected whites do carry one CF gene. Oddly enough. the place to look for an assuredly normal chromosome is in the parents of children with cystic fibrosis. Each biologic parent of a child with CF must have one CF gene and one normal gene. The researchers in Toronto found a three-base pair deletion in a eDNA sample from a library constructed with RNA isolated from epithelial cells cui-
tured from a CF patient's sweat glands. This deletion leads to the loss of a single amino acid (phenylalanine) at position 508 of CFTR (usually referred to as b.F508). This initially seemed likely to be a polymorphism. since there were reasons to expect the CF mutation to be something more gross: a nonsense mutation that would stop translation altogether or a nonconservative missense mutation that would substitute one amino acid for another. But by June 1989, more than 200 CF parents had been studied who had the common mutation on one copy of chromosome 7, and none of them had the same mutation on the other copy (their normal chromosome). At that point. we were confident that we had the CF gene (Figure 3). The data on normal chromosomes now number in the thousands and support our earlier conclusion. Not all mutations responsible for CF are due to b.F508, however. About 25% to 30% of CF chromosomes in whites do not carry b.F508, which indicates that other mutations in CFTR can also be responsible. Intensive efforts from a large consor-
tium of laboratories around the world have so far identified about 50 additional CF mutations, most of which are uncommon in the population.
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Carrier Screening Since one in 20 white North Americans carries the CF gene, effective carrier screening could be clinically important. Within months of the gene's cloning. DNA diagnostic companies and a number of research laboratories were already offering screening to families with a history of cystic fibrosis, where the information can often be of considerable value to the couple at risk (Figure 4). Unfortunately, the tests currently available detect just 70% to 75% of carriers (those with AF508), a level of sensitivity that can cause problems. Despite the potential benefits of carrier screening, careful consideration indicates that the medical profession should not rush into general screening. With testing now available, half of the couples at risk of having a child with cystic fibrosis would be missed. Nor could anyone be assured that he or she does not carry the gene, since 25% to 30% of carriers would be missed by AF508 testing. Because of these uncertainties, an NIH workshop of geneticists, ethicists. and public health officials concluded in March 1990 that across-theboard carrier screening for cystic fibrosis should not yet be considered an applicable standard of medical care. This caution grows in part out of a concern for couples in which one partner tests positive and the other negative. Without screening, the standard risk of a white child's being affected in the absence of a family
history of cystic fibrosis is only one in 2,500. If the parents-tobe are screened for AF508 and one is positive and one negative, suddenly the risk estimated for CF increases to one in 400. Therefore, for couples such as this, who will not be uncommon, the result of screening is likely to be a heightened level of anxiety. In addition, the frequency of the disease and the types of mutations vary according to racial and ethnic backgrounds. Therefore, different laboratory procedures and counseling modifications will be required for different populations. Another major consideration is that the public will need to be better informed about genetics and screening before it can be expected to understand the limitations and uses of an imperfect screening test. Any screening program will have to include a major educational arm, one that can help people make an informed decision about whether they want the test. and offer counseling about the result. For couples who do have a family history of cystic fibrosis on one side or the other, carrier screening is already appropriate. For such couples, the currently available test is likely to give useful information about their odds of having an affected child. Such couples can and should be offered genetic counseling by their primacy physician or through referral to a genetic center. The best time for prospective screening of couples is bEfore conception, when more options are available. For some couples, prenatal diagnosis followed by abortion of an affected fetus is not an acceptable option. Indeed, to what extent that option will be chosen even by those
Figure 4. CF carrier screening now identifies only the most common defect, ~F508, but is useful for couples with a CF family history. A child of two carriers has a 1/4 chance of being affected; an unaffected child has a 2/3 chance of being a carrier. Before testing, the mate of a carrier (assuming Northern European background) has the 1/25 probability of the general population. The probability that both would transmit an abnormality is 1/4, and the likelihood of disease is 1/150 (top). If both parents test positive (middle), the likelihood increases to 1/4. If only the person with a family history tests positive (bottom), it falls but not to zero, because the other partner has a 1/100 chance of carrying an unidentifiable CF defect.
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philosophically comfortable with abortion is unclear. Unlike Tay-Sachs disease and other diseases for which screening was very much welcomed by those at risk, cystic fibrosis is compatible with many years of good-quality life. Median survival now extends to age 26. Whether one talks of prenatal or carrier screening, negative or positive family histories, or tests that are 80%, 90%, or 95% accurate, it is probable that some type of CF screening will eventually be offered. It is equally clear that geneticists, ethicists, and public health officials should take a hard look at the issue of screening. As a negative example, the experience with sickle cell screening should be recalled. Some of those screened for sickle cell trait were not well informed about why the screening was being done and what the results meant. Insurance companies were inappropriately involved and often confused about the difference between being a carrier and having the disease. Such a tragic situation must not be allowed to recur. Pilot projects on well-defined populations, with careful assessment of the outcome, are essential to a successful CF screening program.
1be Basic Defect Although identification of the CF gene did not instantly change clinical medicine, it did immediately focus the research effort. Less than two months after announcement of the gene's cloning, more than 70 laboratories around the world joined forces to explore the gene in a coordinated manner. This unprecedented consortium focuses on the mutations that cause cystic fibrosis. As previously noted, one identified 54
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mutation-the single deleted codon at position 508 (.6.F508) with a consequent loss of a phenylalanine-is found in about 70% of CF chromosomes. Members of the consortium share information about other potentially deleterious mutations well before submission for publication-a remarkable arrangement that prevents pointless duplication of effort and hastens progress toward a better understanding of the disease.
1be CFIR Protein With the gene cloned, we were able to predict the amino acid sequence of the long-elusive CF protein: the cystic fibrosis transmembrane conductance regulator. Of course, the sequence did not tell us everything about what CFTR is doing in the cell. But as long as the gene can, as it does, predict the primary structure of the protein, it raises to a virtual certainty the likelihood that investigators will eventually find the molecular answer to the question, what is cystic fibrosis? Initially, the agenda calls for exploring CITR's role in regulating transport of chloride ions out of epithelial cells. Is CITR directly involved in ion conductance, or does it indirectly regulate that process? To find out. we need a functional assay. We must study CITR in its normal setting (a respiratory epithelial cell or a sweat gland cell), as well as in cells not normally involved with chloride transport. An important step will be to put the protein into cells that do not normally transport chloride ions. Xenopus oocytes are a promising vehicle, since both endogenous and exogenous membrane channels have been well studied in them. Experi-
ments with Xenopus oocytes could help clear up the question of how CITR is involved in the regulation of impermeability. It will also be important to put the normal CITR gene into CF cells to see if the defective cells begin to regulate transport of chloride properly. If this works, it will provide a system for testing the many mutations found in the large CF gene. The goal would be to distinguish between serious defects and harmless polymorphisms. Not long after the gene was cloned, a number of laboratories began trying to make an antibody against CITR. one that could serve as a probe to pinpoint the protein's location in the cell. By examining the CF gene, it is clear that CITR has hydrophobic domains that almost certainly indicate that the protein will be found in cell membranes (Figure 5). This is, of course, no surprise, but with a fluorescently labeled antibody, investigators will be able to locate CITR conclusively. Another important item on the agenda is development of an animal model for cystic fibrosis. With the gene cloned, it should be possible to create such a model and use it to study the biochemistry of CF and to test potential CF therapies. The human CF gene is very similar in sequence to that of the murine counterpart. Using new site-specific recombination technologies, several groups are attempting to develop a mouse strain that carries the f1F508 CF mutation. If such mice prove to have a disease resembling cystic fibrosis, this could provide a valuable model.
Drug Treatment Drug treatment for CF patients is currently limited to
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pancreatic enzyme replacement and to antibiotic therapy for the bacterial infections associated with mucous accretions in the lungs. Except for a recent. promising small trial with aerosolized amiloride, a drug that alters sodium transport. no drugs have been found to positively influence ion transport and prevent the life-threaten-
ing accumulation of mucus. With the CF gene cloned, however, research on treatment can proceed along several lines, some of them even before we have a biochemical understanding of the basic defect. The most straightforward approach would be to produce large amounts of the normal CFTR protein and deliver it to
Cytosol
~
R Domain
Figure 5. The protein encoded by the CF gene has been termed cystic fibrosis transmembrane conductance regulator (CFTR). Its 12 hydrophobic domains appear to anchor the molecule in the plasma membrane, with little of its structure exposed at the cell surface. The portion inside the cell is notable for a large globular region called the R domain and for two smaller regions, nucleotide
affected lung tissues. Since cystic fibrosis is a recessive disease and carriers have 50% abnormal protein but are healthy, we know that the defective protein need not be eliminated. All that is needed is enough normal protein, and in some recessive diseases, as little as 5% normal protein is enough for normal cell function.
ATP Binding 'Domains
~F508 / Mutation
binding folds (NBFs), that are probably capable of binding ATP. The ~F508 defect found in most CF patients (red dot) is in the region of one of the NBFs. With its transmembrane spans and NBFs, the structure of CFTR closely resembles that seen in a family of transport proteins that includes P-glycoprotein, an ATP-binding pump that functions physiologically to rid cells of toxins.
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Although cystic fibrosis is a multiorgan disease, the lungs are the critical target for treatment. It might be possible to aerosolize a suspension of CFfR protein, perhaps inserted into a lipid bilayer that is capable of fusing spontaneously with the cell membrane. How to get the protein into membranes of the right cells (ciliated respiratory epithelial cells) is, however, a major challenge. A trial-and-error search for drugs that promote chloride transport can also proceed in advance of understanding the basic defect. To test the tens of thousands of compounds in stock, a drug company would need an ample supply of the CF version of CFfR and a simple assay for chloride transportperhaps even in an artificial membrane. If this sounds lowtech and inefficient, it is pertinent to recall that most drugs were discovered through trial and error. Rational drug design is still possible, provided progress in understanding the basic defect can be made. If a CF model can be developed and the chloride transport pathway can be dissected, it may be possible to determine what agonists or antagonists promote chloride transport.
Gene 1berapy Gene therapy could be the ultimate intervention. If the normal CFTR gene can be inserted into the right cells, the potential for cure, not just amelioration, could be realized. Having said this, it must be cautioned that gene therapy for cystic fibrosis is unlikely to be available in the near future. But CF gene therapy is a plausible possibility. In many diseases that might benefit from such therapy, gene 56
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delivery is complicated by the affected tissue's inaccessibility. In CF, the airways are relatively accessible, essentially noninvasively. In fact, it has already been shown that retroviruses penetrate rodent respiratory epithelial cells with reasonable efficiency. Retroviruses have often been used to deliver genes because they are efficient at getting into cells and integrating their DNA into the cells' DNA if the cells are dividing. Other viruses may tum out to be better delivery systems. Adenoviruses readily infect the respiratory tract, and much is known about their biology. They have already been used for gene transfer in tissue culture. We know less about respiratory syncytial viruses, but they may also be vector candidates. Conceivably, the best vector may be something like a synthetic lipid vesicle, rather than a virus. Other crucial issues to be addressed before this approach can be successful are the identity of the ideal target cells, the kinetics of cell division, how much protein to make inside the cells, and what proportion of cells need to be transfected in order to improve the patient's health. These questions are beginning to be explored. Much of the preliminary work must be done in animal models. While gene therapy offers great promises, it also poses problems. Clearly, research needs to continue on all fronts simultaneously-gene therapy, drug treatment, and studies of the basic defect.
1be Human Genome Project The success of positional cloning in locating the CF gene confirms something important about the project to identify
human disease genes by mapping and sequencing the human genome: It works. It can be done. A skeptic might point out that the search for this one gene involved tremendous labor and a huge amount of money. What about mapping and sequencing no less than the entire human genome? A large and well-financed collection of laboratories spent more than five years searching for the CF gene, along the way cloning and mapping roughly one part in 6,000 of the human genomeand sequencing only a small fraction of what was cloned. If the entire human genome were to be mapped and sequenced at the same rate, it would take a long, long time. Were the technology for mapping and sequencing the genome static, the human genome project would be in trouble. But the technology is anything but static. Progress has been steady. The yeast artificial chromosome system is one powerful new technique not available during the search for the CF gene. It allows cloning of a region 400 or 500 kb long. It took us almost two years to cross the 300-kb interval between CF markers and the CF gene. Had the yeast artificial chromosome technology been available, the walking and jumping process could have been accelerated. Another new technique, used successfully by one of us (F.S.C.) in the recent identification of the neurofibromatosis gene, might allow the identification of all transcripts in a given region in one experiment instead of many. In this approach, a yeast artificial chromosome is used directly to probe a eDNA library: if successful, all of the transcripts in a 200- to 500-kb region can be
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identified in one experiment rather than via the painstaking, fragment-by-fragment search that was carried out for cystic fibrosis. Mapping and sequencing the human genome's entire 3 billion base pairs is a huge undertaking. Assuming one character per nucleotide, an individual human's haploid genome would fill 13 sets of the Encyclopaedia Brttanntca. Not 13 volumes-13 complete sets. Of course, the project is bigger than any one person's genome. The goal is to be able to find and sequence some of the great variety in the human genomepolymorphisms and mutations alike. And to make sense of this information, it will be necessary concurrently to build maps and sequences of other species. It seems clear that a focused international effort will be more efficient than an ad hoc approach to gathering, storing, and learning to use such a large volume of important data. Some have questioned whether having a complete map and sequence will expedite the process of finding disease genes. One might argue that the genes involved in important diseases, such as cystic fibrosis and Huntington's disease, are going to be found anyway. Many will no doubt be located before the project is advanced enough to aid the search. But there are many harder problems-schizophrenia and Alzheimer's disease. for instance-in which it has been very difficult to locate the responsible gene. In truth, it is hard to imagine current strategies pinpointing the gene for Alzheimer's disease, which in some families seems to be hidden somewhere in a 5 millionto 10 million-base pair section of DNA on chromosome 21 that houses perhaps 100 genes.
The problem is further complicated by the evidence that Alzheimer genes exist on other chromosomes in other families. With a complete map and sequence of the 1 million to 10 million base pairs in the regions of interest. computer analysis could potentially identify most of the coding regions that indicate the presence of genes. One could then look at the DNA sequences of the putative genes to see which code for proteins that look homologous to other proteins in the central nervous system. With the normal sequences of CNS genes already identified as part of the genome project, the relevant DNA of Alzheimer's patients could be much more readily screened for mutations. In diseases that involve several genes, the mapping and precise localization of responsible loci are certain to be more difficult even than problems postulated for Alzheimer's disease. Thus, when the genetic basis of disorders such as hypertension, coronary artery disease,
and breast cancer is pursued, there is not likely to be much success until considerable map information is available. Another crucial need of this effort is to collect DNA from many families with the diseases to be studied. A detailed map and complete sequence of normal, healthy genes will not, by themselves, reveal very much about diseases. The genetic analysis that points the way to disease genes depends on a large number of DNA samples from affected families. In summary, the search for the CF gene has taught us much about the positional cloning strategy, and it is likely that many other genes for human disorders will be found in this fashion, especially with the advances expected from the human genome project. The current challenge in CF research is to use the newly found knowledge about CITR to deduce the basic biochemical defect, and to develop an effective treatment for those with the disease. D
Selected Reading Rommens JM et al: Identification of the cystic fibrosis gene: Chromosome walking and jumping. Science 245: 1059. 1989 Riordan JR et al: Identification of the cystic fibrosis gene: Cloning and characterization of complementaiy DNA Science 245: 1066. 1989 Kerem B et al: Identification of the cystic fibrosis gene: Genetic analysis. Science 245:1073. 1989 Iannuzzi MC. Collins FS: Update: Reverse genetics and cystic fibrosis. Am J Respir Cell Mol Bioi 2:309. 1990 Lemna WK et al: Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis. N Engl J Med 322:291. 1990 Statement from the NIH Workshop on Population Screening for the Cystic Fibrosis Gene. N Engl J Med 323:70. 1990 Orkin S: Reverse genetics and human disease. Cell 4 7:845, 1986 WhiteR, Lalouel J-M: Chromosome mapping with DNA markers. Sci Am 258:40, Februacy 1988 McKusick VA: Mapping and sequencing the human genome. N Engl J Med 320:910, 1989 Watson JD: The human genome project: Past, present, and future. Science 248:44, 1990
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